High-speed on demand droplet generation and single cell encapsulation driven by induced cavitation

ABSTRACT

Methods and devices for the formation of droplets of a first fluid in a second fluid and the encapsulation of particles or cells within such droplets are disclosed. Impetus for droplet formation is provided by the creation of a transient bubble, which may be induced using a pulsed laser. Droplet volume and the frequency at which droplets are formed can be controlled by modulation of the pulsed laser. The disclosed methods and devices are particularly suitable for use in microfluidic devices.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/370,196, filed on Feb. 9, 2012, issued as U.S. Pat. No. 9,176,504which claims benefit of and priority to USSN 61/442,009, filed on Feb.11, 2011, both of which are incorporated herein by reference in theirentirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Grant NumberEY018228, awarded by the National Institutes of Health, and GrantNumbers 0747950, 0852701, and 0901154, awarded by the National ScienceFoundation. The government has certain rights in the invention.

The invention described herein was made in the performance of work underNASA cooperative agreement NCC2-1364, and is subject to the provisionsof Public Law 96 -517 (35 USC 202 ) in which the Contractor has electedto retain title.

FIELD

The present invention relates to the field of microfluidics. In certainembodiments methods and devices are provided for the high speedformation of droplets and/or the encapsulation of droplets, particles,and/or cells.

BACKGROUND

Microfluidic devices have attracted great interest as they to provide aplatform for performing analyses on extremely small volumes of fluid,and when produced utilizing photolithography techniques can bemanufactured inexpensively. These devices have the potential to act as a“lab on a chip”, integrating multiple functionalities including, forexample, sample preparation, thermal cycling to support the polymerasechain reaction, and absorbance or fluorescence monitoring. Their compactsize makes them particularly suitable for use in portable devices,potentially allowing the performance of sophisticated analyses in aclinician's office or in the field. One of the challenges with usingmicrofluidic devices in the analysis of multiple samples, however, issample compartmentalization. While a conventional laboratory analyzermay utilize a series of cuvettes or similar receptacles to preventcontamination between samples this approach is difficult to implementwith small volumes of fluid, where interactions with device surfaces cansupersede bulk flow properties.

Typical microfluidic devices utilize a single fluid phase that flowscontinuously through the device. Introduction of a discrete volume offluid test sample or reagent into such a device leads to the formationof a fluid segment that moves through the channels of the apparatus.Unfortunately, such a fluid segment will tend to become dispersed due toforces such as diffusion and turbulence within the flow channel. Inaddition it is possible for components of the fluid segment to interactwith the walls of channels of the microfluidic device, only to bereleased at a later time. Such phenomena can result in contaminationbetween fluid segments and results in the need to design suchmicrofluidic chips with features to reduce turbulence within fluidchannels and to design test protocols that incorporate time consumingwashing or flushing of the interior volume between samples. In addition,dispersion of fluid segments makes it difficult to provide reproduciblevolumes and concentrations of fluid segment contents forcharacterization reactions.

One approach to resolving this issue has been the introduction ofdigital microfluidic devices, in which sample fluids for analysis orother treatment are introduced into the channels of the device in theform of discrete, low volume droplets. For example, introducing aqueoussamples with biochemical or biological contents as aqueous droplets thattravel within a channel containing an immiscible oil medium reducesinteraction with the channel wall and prevents dispersion, minimizingcontamination between droplets. Reagents used in the characterization ofsuch samples can be treated similarly. In order to be effective,however, a digital microfluidic device requires a mechanism forhigh-speed droplet generation with precise volume control in order tofully realize accurate, high throughput analysis.

Passive mechanisms may be used for rapid, continuous droplet generationas a function of flow through such a device. Highly uniform droplets canbe generated at a rate of thousands of drops per second in this fashion(Yobas et al. (2006) Lab on a Chip, 6:1073-1079). U.S. Pat. No.7,759,111 describes such a device, where droplets are sheared from astream of aqueous media by a flow of immiscible oil. Another example ofa passive device is disclosed in WO 2010/110843A1, in which a barrierintruding into a fluid channel acts in combination with fluid and flowcharacteristics of the channel to form vortices that provide periodicvariations in pressure that drive droplet formation. Such devices,however, do not provide on demand generation of a droplet containingspecifically designated volume of sample fluid (for example, a volumecontaining a particular cell of interest) and do not lend themselves tothe production of individual droplets with different volumes. Thislimits their utility for the characterization of different samplesvolumes and in the performance of a variety of testing protocols.

Active methods for droplet generation, which rely on the use of anapplied force to drive droplet formation, can address these issues. Suchdevices may incorporate physical components that regulate flow throughthe device. One example of this is the use of pneumatically drivenmicrovalves that are integrated into the microfluidic device (Zeng etal. (2009) Lab on a Chip 9:134-1343), which permitted controlled dropletformation at rates as high as 100 droplets per second. Another exampleof this approach is the use of a movable wall of flexible material(PDMS) that is integrated into the microfluidic chip and driven by airpressure to periodically interrupt the flow of a fluid phase in order toprovide a dispersion (Hsiung et al. (2006) J. Micromechanics andMicroengineering, 16: 2403-2410), which demonstrated rates of dropletformation as high as 20 per second. Yet another example, US2010/0059120, discloses the use of a two channels connected by anopening, in which a flow interruptor in one channel can be triggered toblock fluid flow and force a portion of its contents into the secondchannel. Another example of such a device is described in US2010/0163412, which discloses a device that incorporates a flexiblefluid reservoir that is compressed briefly by an imbedded piezoelectricdevice to generate pressure for droplet formation. Such features addsignificantly complexity to the design of these microfluidic devices,further complicating the manufacturing process. The mechanical nature ofsuch approaches limits the frequency at which droplets can be producedand may show changes in performance over time. In addition, theseapproaches tend to produces droplet populations with greater variationin droplet size than those produced using passive devices.

Other approaches to active droplet generation have relied on the use ofmassless or essentially massless energies applied to the device in orderto avoid the disadvantages of mechanical components. Some of theseutilize the application of electrical fields to the device to alterfluid flow or change the properties of the interface between two fluidsin order to facilitate droplet formation. This can require largedifferences in conductivity between the fluids involved, which limitsthe utility of such devices. For example, US 2006/0231398 discloses theuse of potential differences to move droplets between immiscible low andhigh resistance fluids by electrowetting, utilizing a potentialdifference to temporarily lower the surface tension at the interfacebetween the fluids until the existing flow pattern is sufficient togenerate droplets. A similar approach is described in WO 2010/151776, inwhich a potential difference drives a combination of effects, includingelectrokinetic flow and interference in the interface between twoimmiscible fluids, to generate droplets. Yet another example of the useof potential differences to drive droplet formation is found in WO2011/023405A1, which discloses a combination of a nozzle structure andestablishment of a potential difference to electrospray droplets of aconductive fluid into a fluid dielectric. An approach that does notrequire large conductivity differences between the fluids involved indroplet formation is disclosed in US 2005/0031657, which describesheating a portion of a container within the device using a resistanceheater until a portion of the fluid stored therein is vaporized.Pressure from the vaporized fluid pushes a portion of remaining fluidthrough a nozzle into an immiscible fluid. Droplet generation from thisapproach is relatively slow, however, producing only around 15-25droplets per second per nozzle. While these approaches avoid the use ofmechanical components, they require the incorporation of electrodes,resistance heaters, or similar components into the device. This addscomplexity to the design of the device and further requires the use ofsupporting features for reliably supplying current.

SUMMARY

In various embodiments novel methods and devices for rapidly andreproducibly generating droplets of a first fluid in a second fluid aredescribed herein. The fluids may be immiscible, where the immisciblefluids can include fluids that are not significantly soluble in oneanother, fluids that do not mix for a period of time due to physicalproperties such as density or viscosity, and fluids that do not mix forperiods of time due to laminar flow. Droplet formation is driven by theexpansion and subsequent contraction of transient bubbles (such ascavitation bubbles) within the first fluid. Alternatively, the bubbleformation within a first fluid may cause it to act on a second fluidthereby driving generation of droplets of the second fluid in a thirdfluid. Cavitation bubbles can be generated using a directed energysource, thereby removing the need to incorporate electrodes, heaters, orsimilar components into devices incorporating the invention. Suitabledirected energy sources include, but are not limited to, a pulse laser,use of which permits on demand formation of highly reproducible dropletsat speeds from less than 1 up to 100,000 droplets per second. Dropletvolume can be controlled, with droplet volumes, in certain embodimentsranging from about 1 to about 150 picoliters. In certain embodimentslive cells can be captured within such droplets, with high cellviability, in certain embodiments of up to 92.07%. Since mechanicalvalves or pumps are not needed these methods and devices areparticularly suitable for use in microfluidic devices.

In one embodiment a first fluid and a second fluid, which may beimmiscible, are operatively coupled. In certain embodiments theoperative coupling can take the form of a fluid communication. In otherembodiments a flexible membrane may be interposed between the firstfluid and the second fluid. Generation of a cavitation bubble within thefirst fluid generates sufficient velocity and/or impulse and/ordisplacement to the first fluid to move a controlled volume of thesecond fluid. In certain embodiments such a cavitation bubble expandsand contracts within 1 millisecond, can move a controlled volume ofabout 1 microliter or less. Such cavitation bubbles may be produced byirradiation, for example by a pulse laser. In some embodiments thevolume of the controlled volume of the second fluid can be controlled bythe energy and/or pulse frequency, and/or wavelength of the pulse laser,which in turn may be modulated by a controller.

In another embodiment a first fluid path and a second fluid path arecoupled via an opening. In some embodiments fluids in the first andsecond fluid paths are immiscible. Generation of a cavitation bubblewithin the first fluid path imparts sufficient velocity to a portion ofthe first fluid to cause a droplet of the first fluid to move across theopening and into the second fluid path. In certain embodiments theopening may be configured as a port, a channel, or nozzle. Suchcavitation bubbles may be produced by irradiation, for example by apulse laser. In some embodiments intensity, duration, and/or position ofthe laser irradiation can be modulated the volume of the droplet.

In another embodiment of the invention a first fluid path and a secondfluid path are in fluid communication, and the second fluid path iscoupled to a third fluid path via an opening. In some embodiments fluidsin the second and third fluid paths are immiscible. Generation of acavitation bubble within the first fluid path imparts sufficientvelocity to a portion of the second fluid to cause a droplet of thesecond fluid to move through the opening and into the third fluid path.In certain embodiments the opening may be configured as a port, achannel, or nozzle. In some embodiments the second fluid may includeparticles and/or cells. The second fluid path may be monitored, withdata produced by such monitoring being transmitted to a controller. Insome embodiments cavitation bubbles are produced by irradiation, whichmay be initiated by a controller. Irradiation can be in the form of alaser pulse, and in some embodiments the volume of the droplet may bemodulated using the intensity, duration, and position of the laserpulse.

In another embodiment a flexible membrane is interposed between a firstfluid path and a second fluid path, and the second fluid path is coupledto a third fluid path via an opening. In some embodiments fluids in thesecond and third fluid paths are immiscible. Generation of a cavitationbubble within the first fluid path results in the elastic deformation ofa portion of the flexible membrane into the second fluid path. Thiselastic deformation imparts sufficient velocity to a portion of thesecond fluid to cause a droplet of the second fluid to move through theopening and into the third fluid path. The opening may be configured asa controller. In some embodiments the second fluid may include particlesand/or cells. The second fluid path may be monitored, with data producedby such monitoring being transmitted to a controller. In someembodiments cavitation bubbles are produced by irradiation, which may beinitiated by a controller. Irradiation can be in the form of a laserpulse, and in some embodiments the volume of the droplet may bemodulated using the intensity, duration, and position of the laserpulse.

In another embodiment of the invention a first fluid path and a secondfluid path are connected by an opening, where the opening is positionedsuch that formation of a bubble in the first fluid path can induce aforce that causes a droplet of the first fluid to move through theopening and into the second fluid path. In certain embodiments theopening may be configured as a port, a channel, or nozzle. In someembodiments the opening is configured as a nozzle. A controller iscoupled to an energy source (such as, for example, a pulse laser) thatcan direct energy into the first fluid path to cause the formation ofone or more bubbles. In some embodiments the bubble can be a cavitationbubble. In still other embodiments the energy source is a pulse laser;the controller may be configured to adjust the volume of the droplet bymodulating the intensity, duration, and/or position of a laser pulseproduced by a pulse laser.

In still another embodiment a first fluid path and second fluid path arepositioned such that a flexible membrane is interposed between them. Theflexible membrane is in turn positioned such that formation of a bubblewithin the first fluid path results in the elastic deformation of aportion of the flexible membrane, which in turn induces a force on fluidcontained in the second fluid path. The second fluid path and a thirdfluid path are connected by an opening, which is disposed such that whensuch a force is exerted on the fluid of the second fluid path a dropletof the fluid is extruded through the opening into the third fluid path.A controller is configured to direct energy that results in thetemporary formation of a bubble in the first fluid path. This temporarybubble can be a cavitation bubble. In certain embodiments the openingmay be configured as a port, a channel, or nozzle. In some embodimentsthe opening is configured as a nozzle. In various embodiments a monitormay be configured to monitor the second fluid path or the third fluidpath, where the monitor transmits the data gathered to the controller.In certain embodiments the controller may be configured to control thetransfer of a designated volume of the fluid in the second fluid pathinto the third fluid path, where the designated volume is determinedusing data from the monitor.

In some embodiments a fluid path may include particles or cells that areencapsulated by droplets of the surrounding fluid that are extruded intoanother fluid path. A monitor may be included in some embodiments tocharacterize such particles or cells which, when coupled with acontroller, may permit controlled encapsulation of specific particles orcells within a specified droplet.

In various embodiments devices for the generation of droplets areprovided. In certain embodiments the device comprises a first fluidstream (e.g., microfluid stream) comprising a first fluid adjacent to asecond fluid stream (e.g., microfluid stream) comprising a second fluidwhere the second fluid is immiscible in the first fluid. In certainembodiments the device comprises a first microfluidic channelcomprising, containing and/or directing the first fluid stream; and asecond microfluidic channel comprising, containing and/or directing thesecond fluid stream where the first microfluidic channel is adjacent to,or in proximity to, the first microfluidic channel and is in fluidcommunication with the second channel (e.g., via a port or a channel).In certain embodiments the second fluid comprises an aqueous fluid. Incertain embodiments the first fluid comprises an oil or an organicsolvent. In certain embodiments the first fluid comprises a solventselected from the group consisting of carbon tetrachloride, chloroform,cyclohexane, 1,2-dichloroethane, dichloromethane, diethyl ether,dimethyl formamide, ethyl acetate, heptane, hexane, methyl-tert-butylether, pentane, toluene, and 2,2,4-trimethylpentane. In certainembodiments the first fluid comprises an oil. In certain embodiments thedevice comprises a third fluid stream disposed between the firstmicrofluid stream and the second microfluid stream. In certainembodiments the device comprises a third fluid stream disposed betweenthe second fluid and the port or channel. In certain embodiments thedevice further comprises a third fluid stream disposed in the secondmicrofluidic channel between the port and the second fluid. In certainembodiments the third fluid stream contains droplets, cells, orparticles that are to be encapsulated. In certain embodiments the portor channel comprises a nozzle. In certain embodiments the first and/orsecond microfluidic channel is formed from a material selected from thegroup consisting of glass, metal, ceramic, mineral, plastic, andpolymer. In certain embodiments the first and/or second microfluidicchannel is formed from an elastomeric material (e.g.,polydimethylsiloxane (PDMS), polyolefin plastomers (POPs),perfluoropolyethylene (a-PFPE), polyurethane, polyimides, andcross-linked NOVOLAC® (phenol formaldehyde polymer) resin, and thelike).

In certain embodiments the device produces a substantially continuousvolume tuning of droplet ranging from about 0.1 fL or about 1 fL, orabout 10 fL or about 50 fL, or about 100 fL, or about 500 fL up to about1 μL, or about 500 nL, or about 1 nL, or about 500 pL, or about 400 pLor about 300 pL or about 200 pL or about 150 pL. In certain embodimentsthe device produces a substantially continuous volume tuning of dropletranging from about 0.1 fL to about 1 μL, or about 0.1 fL up to about 500nL, or about 1 fL up to about 1 nL, or about 1 fL up to about 500 pL, orabout 500 fL up to about 500 pL or about 1 pL up to about 150 pL. Incertain embodiments the device can provide on-demand droplet generationat a speed of greater than about 1,000, more preferably greater thanabout 2,000 droplets/sec, more preferably greater than about 4,000droplets/sec, more preferably greater than about 6,000 droplets/sec, ormore preferably greater than about 8,000 droplets/sec. In certainembodiments the device can provide on-demand droplet generation at aspeed ranging from zero droplets/sec, 1 droplets/sec, 2 droplets/sec,about 5 droplets/sec, about 10 droplets/sec, about 20 droplets/sec,about 50 droplets/sec, about 100 droplets/sec, about 500 droplets/sec,or about 1000 droplets/sec, up to about 1,500 droplets/sec, about 2,000droplets/sec, about 4,000 droplets/sec, about 6,000 droplets/sec, about8,000 droplets/sec, about 10,000 droplets/sec, about 20,000droplets/sec, about 50,000 droplets/sec, or about 100,000 droplets/sec.In certain embodiments the device can provide on-demand dropletgeneration at a speed of greater than about 1,000, more preferablygreater than about 10,000, more preferably greater than about 20,000droplets/sec, more preferably greater than about 40,000, more preferablygreater than about 50,000 droplets/sec, more preferably greater thanabout 80,000, or more preferably greater than about 100,000droplets/sec. In certain embodiments the device is present in (or acomponent of) a system comprising an energy source capable of forming abubble in a fluid stream or a microchannel. In certain embodiments theenergy source comprises an optical energy source or microwave emitter.In certain embodiments the energy source comprises a laser (e.g., apulse laser). In certain embodiments the device and/or system areconfigured to excite vapor bubbles in the second microfluidic stream. Incertain embodiments the device and/or system are configured to excitevapor bubbles in the second microfluidic channel in proximity to theport or channel. In certain embodiments the device and/or system areconfigured to excite vapor bubbles in a third microfluidic channel orchamber that is not in fluid communication with the first or secondmicrofluidic stream. In certain embodiments the vapor bubbles areexcited in a liquid or gel medium. In certain embodiments the wherevapor bubbles are excited in an oil or non-aqueous medium. In certainembodiments the vapor bubbles are excited in a medium that compriseslight-absorbing nano/microparticles (e.g. dye molecules, metalnanoparticles, and the like). In certain embodiments the device isdisposed on a substrate comprising a material selected from the groupconsisting of a polymer, a plastic, a glass, quartz, a dielectricmaterial, a semiconductor, silicon, germanium, ceramic, and a metal ormetal alloy. In certain embodiments the device is integrated with othermicrofluidic components (e.g., microfluidic components such as PDMSchannels, wells, valves, and the like). In certain embodiments thedevice is a component of a lab-on-a-chip.

In various embodiments systems are provided for the generation ofdroplets and/or the encapsulation of particles or cells. In certainembodiments the systems a device as described above (or below), and anexcitation source for forming gas bubbles in a fluid. In certainembodiments the excitation source is a laser, a microwave source, or anultrasonic energy source. In certain embodiments the system furthercomprises components for detecting particles or cells in the system(e.g., an optical detection system, an electrical detection system, amagnetic detection system, an acoustic wave detection system, anelectrochemical detection system, and the like). In certain embodimentsthe components comprise an optical detection system for detectingscattering, fluorescence, or s ramen spectroscopy signal.

In various embodiments methods for generating droplets are provided. Incertain embodiments the methods involve providing a device as describedabove (and below herein); and utilizing an energy source to form bubblesin the second microfluidic stream or the second microfluidic channel orin a third microfluidic channel or chamber to inject droplets into thefirst microfluidic stream or channel. In certain embodiments theutilizing an energy source comprises utilizing a pulse laser to excitecavitation bubbles in the second microfluidic stream or channel or inthe third microfluidic channel or chamber.

In various embodiments methods of moving a controlled amount of a fluidare provided. In certain embodiments such methods comprise: generating acavitation bubble in a first fluid, where the cavitation bubble impartsa sufficient velocity to a portion of the first fluid to thereby move acontrolled volume of a second fluid that is operatively coupled to thefirst fluid. In certain embodiments the controlled volume of the secondfluid is about 10 μL or less, or about 5 μL or less, or about 1 μL orless, or about 500 nL or less, or about 100 nL or less, or about 1 nL orless, or about 500 pL or less, or about 200 pL or less. In certainembodiments the cavitation bubble has a duration about 100 ms or less,or about 50 ms or less, or about 1 ms or less, or about 0.5 ms or less,or about 1 ms or less or about 0.5 ms or less, or about 0.1 ms or less,or about 0.05 ms or less. In certain embodiments the controlled volumeof the second fluid is 1 μL or less and the duration of the cavitationbubble is about 1 ms or less. In certain embodiments the first fluid andthe second fluid are in fluid communication. In certain embodiments aflexible membrane is interposed between the first fluid and the secondfluid. In certain embodiments the first and second fluids areimmiscible. In certain embodiments the cavitation bubble is generated byirradiation of a volume of the first fluid with a pulsed laser. Incertain embodiments the method further comprises controlling thecontrolled volume of the second fluid using a controller that adjusts atleast one of energy and/or pulse frequency, and/or wavelength of thepulsed laser. In certain embodiments the method comprises generating aplurality of separate and additional cavitation bubbles at a frequencyof at least about 1000 Hz, or at least about 5,000 Hz, or at least about10,000 Hz. In certain embodiments the controlled volume of the secondfluid is about 500 nanoliters or less. In certain embodiments thecontrolled volume of the second fluid is about 200 pL or less. Incertain embodiments the method is repeated at a frequency of about 1 kHzor greater, or at a frequency of about 5 kHz or greater, or at afrequency of about 10 kHz or greater.

In various embodiments methods for generating droplets in a device areprovided. In certain embodiments the methods comprise: providing a firstfluid path comprising a first fluid; a second fluid path comprising asecond fluid; and an opening fluidly coupling the first fluid path tothe second fluid path; and generating a cavitation bubble in the firstfluid path, where the cavitation bubble imparts sufficient velocityand/or impulse and/or displacement to a portion of the first fluid so asto extrude a droplet of the first fluid across the opening and into thesecond fluid path. In certain embodiments the first fluid and the secondfluid are immiscible fluids. In certain embodiments the first fluid isan aqueous fluid and the second fluid is an organic solvent or an oil.In certain embodiments the second fluid is an aqueous fluid and thefirst fluid is an organic solvent or an oil. In certain embodiments theopening is configured as a nozzle. In certain embodiments the cavitationbubble is generated by irradiation of a volume of the first fluid with apulsed laser. In certain embodiments the method involves selecting atleast one of an intensity, duration, wavelength and position of thelaser pulse to thereby produce a desired volume of the droplet.

In certain embodiments methods for generating droplets in a device areprovided comprising: providing a first fluid path comprising a firstfluid; a second fluid path comprising a second fluid, the second fluidpath in fluid communication with the first fluid path; a third fluidpath comprising a third fluid; and an opening fluidly coupling thesecond fluid path to the third fluid path; and generating a cavitationbubble in the first fluid path, where the cavitation bubble impartssufficient velocity to a portion of the second fluid to extrude adroplet of the second fluid across the opening and into the third fluidpath. In certain embodiments the second fluid and the third fluid areimmiscible fluids. In certain embodiments the second fluid is an aqueousfluid and the third fluid is an organic solvent or an oil. In certainembodiments the second fluid is an aqueous fluid and the third fluid isan organic solvent or an oil. In certain embodiments the the methodfurther comprises monitoring the second fluid path and transmitting datagenerated by such monitoring to a controller. In certain embodiments thesecond fluid further comprises a particle. In certain embodiments thesecond fluid further comprises a cell. In certain embodiments theopening is configured as a nozzle. In certain embodiments the cavitationbubble is generated by irradiation of a volume of the first fluid. Incertain embodiments the irradiation is initiated by a controller. Incertain embodiments the irradiation is a laser pulse. In certainembodiments the method further comprises selecting at least one of anintensity, duration, wavelength, and position of the laser pulse tothereby produce a desired volume of the droplet.

In certain embodiments methods for generating droplets in a device areprovided comprising: providing a first fluid path comprising a firstfluid, a second fluid path comprising a second fluid, a third fluid pathcomprising a third fluid, a flexible membrane interposed between thefirst fluid path and the second fluid path, and an opening between thesecond fluid path and the third fluid path; and generating a cavitationbubble in the first fluid path that elastically deforms a portion of theflexible membrane (e.g., a membrane fabricated form an elastomericmaterial (e.g. polydimethylsiloxane (PDMS), polyolefin plastomers(POPs), perfluoropolyethylene (a-PFPE), polyurethane, polyimides, andcross-linked NOVOLAC® (phenol formaldehyde polymer) resin, and thelike)) into the second fluid path, where the elastic deformation of theportion of the flexible membrane imparts sufficient velocity and/orimpulse, and/or displacement to a portion of the second fluid to extrudea droplet of the second fluid across the opening and into the thirdfluid path. In certain embodiments the second fluid and the third fluidare immiscible fluids. In certain embodiments the second fluid is anaqueous fluid and the third fluid is an organic solvent or an oil. Incertain embodiments the second fluid is an aqueous fluid and the thirdfluid is an organic solvent or an oil. In certain embodiments the methodfurther comprises monitoring the fluid in the second fluid path andtransmitting data generated by such monitoring to a controller.

In various embodiments devices for generating droplets are provided. Incertain embodiments the devices comprise a first fluid path; a secondfluid path; an opening between the first fluid path and the second fluidpath, the opening disposed such that formation of a bubble in a fluid inthe first fluid path induces a force in the in an amount effective tothereby extrude a droplet of the fluid from the first fluid path throughthe opening into the second fluid path; and a controller coupled to anenergy source that is and operatively configured to cause the energysource to direct an energy that induces temporary formation of one ormore bubbles in the first fluid path. In certain embodiments the bubbleis a cavitation bubble. In certain embodiments the opening is configuredas a nozzle. In certain embodiments the energy source a pulsed laser. Incertain embodiments the controller is configured to adjust volume of thedroplet as a function of at least one of an intensity of the laserpulse, a duration of the laser pulse, a wavelength of the laser pulse,and a position of the laser pulse within the first fluid channel.

In various embodiments devices for generating droplets are provided. Incertain embodiments the devices comprise a first fluid path; a secondfluid path; a third fluid path; a flexible membrane interposed betweenthe first fluid path and the second fluid path, the flexible membranedisposed such that formation of a bubble in a fluid in the first fluidpath induces a force that elastically deforms a portion of the flexiblemembrane; an opening between the second fluid path and the third fluidpath, the opening disposed such that elastic deformation of a portion ofthe flexible membrane induces a force on a second fluid to therebyextrude a droplet of the second fluid from the second fluid path throughthe opening into the third fluid path; and a controller operativelyconfigured to direct an energy that induces temporary formation of thebubble in the fluid in the first fluid path. In certain embodiments thebubble is a cavitation bubble. In certain embodiments the device furthercomprises a monitor configured to monitor the second or third fluidpath, and further configured to transfer data from the monitor to thecontroller. In certain embodiments the controller is further configuredto control a designated volume of the second fluid into the third fluidpath, the designated volume being determined at least in part by datafrom the monitor.

In certain embodiments of any of the foregoing methods and devices,droplets are generated with droplet volume variations of about 10% orless, preferably about 5% or less, more preferably about 3% or less, orabout 2% or less, or about 1% or less at repetition rates ranging fromabout 1 kHz up to about 10 kHz.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depicts the process of creating a cavitation bubbleusing a pulsed laser. FIG. 1A shows plasma generation within a volume offluid as a result of irradiation with a focused laser pulse, followed bygeneration of a shockwave and cavitation bubble expansion and subsequentcollapse. FIG. 1B shows a graph of a typical time course for expansionand subsequent collapse of a cavitation bubble.

FIG. 2 illustrates the time course of formation and subsequent collapseof a cavitation bubble generated using a pulsed laser.

FIG. 3 schematically illustrates generation of droplets within a fluidchannel in accordance with one embodiment of the invention.

FIG. 4 schematically illustrates generation of droplets that incorporateparticles or cells within a fluid channel in accordance with anotherembodiment of the invention.

FIG. 5 schematically illustrates generation of droplets within a fluidchannel in accordance with another embodiment of the invention.

FIG. 6 schematically illustrates generation of droplets within a fluidchannel in accordance with another embodiment of the invention.

FIG. 7 schematically illustrates generation of droplets that incorporateparticles or cells within a fluid channel in accordance with anotherembodiment of the invention.

FIG. 8, panels (a)-(i), show a time-resolved image series of on-demanddroplet generation.

FIG. 9 depicts modulation of the volume of a generated droplet byvarying the energy of the laser pulse and by varying the location of thelaser pulse.

FIG. 10, panels (a)-(d), illustrate continuous generation of dropletswithin a fluid channel using a series of laser pulses repeated atdifferent intervals. The scale bar has a length of 100 microns. Panel(a) illustrates 2 millisecond intervals, panel (b) illustrates 1millisecond intervals, panel (c) illustrates 500 microsecond intervals,and panel (d) illustrates 100 microsecond intervals.

FIG. 11, panels a and b, illustrate collected droplets generated atdifferent laser pulse frequencies. The scale bar has a length of 100microns. Panel (a) illustrates droplets generated by a laser pulsefrequency of 1 kHz. Panel (b) illustrates droplets generated by a laserpulse frequency of 10 kHz.

FIG. 12, panels a-d, illustrates continuous generation of dropletswithin a fluid channel using a series of laser pulses repeated atdifferent intervals. Panel (a) illustrates 2 millisecond intervals,panel (b) illustrates 1 millisecond intervals, panel (c) illustrates 500microsecond intervals, and panel (d) illustrates 100 microsecondintervals.

FIG. 13, panels a-c, depict encapsulation of a particle or cell within adroplet generated by a cavitation bubble. Panel (a) shows particleswithin a fluid channel. Panel (b) shows the position of a cell,indicated by a white arrow, before and at different time intervalsfollowing the induction of a cavitation bubble and subsequent generationof a droplet. Panel (c) illustrates continuous generation of series ofdroplets that each encapsulate a cell.

FIG. 14 illustrates consecutive generation of droplets within a fluidchannel using laser pulses at a frequency of 1 Hz.

DETAILED DESCRIPTION

In various embodiments devices and methods are provided for on-demandhigh speed droplet generation of droplets of controlled volume that ha eparticular application in the field of microfluidics. In variousembodiments, the methods and devices can also be used to encapsulatecells and/or particles, and/or other fluid droplets.

In various embodiments the devices and methods described herein utilizea novel controllable actuation mechanism, utilizing directed energy thatinduces short-lived cavitation bubbles. In some embodiments this energyis in the form of a pulse laser that provides bursts of optical energy,the intensity, duration, wavelength, and/or position of which can becontrolled.

FIG. 1A illustrates the underlying mechanism of laser pulse inducedcavitation bubble formation in aqueous media. A laser pulse is focusedon a specified volume of the aqueous medium. Absorption of this opticalenergy results in a breakdown of water molecules within the area offocus, generating a plasma bubble near the focal point. The componentsof the plasma recombine in a few nanoseconds, generating a shockwave ofreleased energy and an explosive vapor bubble (also referred to as acavitation bubble) that expands as rapidly as 100 meters per secondfollowed by a rapid collapse. FIG. 1B shows a typical time course forbubble formation and collapse. Bubble radius can be seen to increaserapidly up to approximately 1 microsecond following initiation, followedby a rapid collapse.

FIG. 2 shows a series of photomicrographs of bubble formation using thisactuation mechanism. A shockwave can be seen expanding outwards from thepoint of plasma generation at 22 nanoseconds following initiation. Arapidly expanding cavitation bubble is readily observable at 72nanoseconds, with the bubble expanding out of the frame by 55microseconds. This is followed by a rapid collapse of the bubble, whichis essentially complete by 152 microseconds following initiation. Thepressure inside such a bubble can be as high as tens of megapascals ormore as the bubble expands. A number of unique properties, such as rapidactuation of the driving force (femtoseconds to nanoseconds, dependingon laser pulse duration), rapid conversion of the directed energy intomechanical power, the large magnitude of the resulting forces, therelatively large displacement produced by the cavitation bubble, and theextremely transient nature of the forces involved provide a uniquemechanism for ultrafast micro- and nano-fluidic actuation. Utilizingthis actuation mechanism, micro- and nano-fluidic components such asswitches, valves, and pumps can be realized to guide, drive, andregulate fluid flows at micro- and nano-fluidic scales withunprecedented speed and accuracy, thereby enabling novelfunctionalities.

One illustrative embodiment of the invention is shown schematically inFIG. 3. The figure shows a device, which can be a microfluidic device,comprising a first fluid channel (320) (e.g., a microchannel) containinga first fluid (312) and a second fluid channel (310) (e.g., amicrochannel) containing a second fluid (322) where the second fluid isimmiscible in the first fluid and where the fluid channels are in fluidcommunication with each other via an opening (330). In some embodimentsthis opening is in the form of a nozzle. A directed energy source (340),for example a pulse laser, is directed towards the first fluid channel(320). In certain embodiments, the laser can be directed using, forexample, a mirror (350) and focused into a volume of the first fluidchannel (320) using a lens (355). In some embodiments the mirror and/orthe lens are configured to permit focusing of the directed energy sourceat different positions within the first fluid channel (320). Thedirected energy source (340) initiates the formation of a transientbubble (360) (e.g., a cavitation bubble) within the first fluid channel(320), driving a droplet of the first fluid (370) into the second fluidchannel (310). Collapse of the bubble causes a back flow of the extrudedfirst fluid, causing the formation of a narrow “neck” and quicklyleading to the release the droplet (380) into the second fluid channel(310).

A series of photographs showing the formation and release of a dropletin such a device is shown in FIG. 8. FIG. 8, panel (a), shows a set ofparallel fluid channels connected by an opening. Induction of acavitation bubble is seen in FIG. 8, panel (b), which extrudes a portionof the contents of one channel into the other as can be seen in FIG. 8,panels (c) to (e). As the bubble collapses a narrow “neck” of connectingfluid is formed, as seen in FIG. 8, panels (f) and (g). Finally, thisneck retracts and the droplet is released as shown in FIG. 8, panels (h)and (i).

Another embodiment of the invention is shown in FIG. 4. The figure showsa device, which can be a microfluidic device, comprising a first fluidchannel (420) (e.g., a microchannel) containing a first fluid, a secondfluid channel (415) (e.g., a microchannel) containing a second fluid(417), and a third fluid channel (410) (e.g., a microchannel) containinga third fluid (412) where the second fluid is immiscible in the thirdfluid and where the second fluid channel and the third fluid channel arein fluid communication with each other via an opening (430). In someembodiments this opening is in the form of a nozzle. In certainembodiments the second fluid may include particles or cells (416), andcan be immiscible in the first fluid by virtue of laminar flow and/or byvirtue of chemical immiscibility. A directed energy source (440), forexample a pulse laser, is directed towards the first fluid channel(420), optionally using a mirror (450) and directed, and optionallyfocused, into a volume of the first fluid channel (420) using, forexample, a lens (455). In some embodiments the mirror and/or the lensare configured to permit focusing of the directed energy source atdifferent positions within the first fluid channel (420). The directedenergy source (440) initiates the formation of a transient bubble (460)(e.g., a cavitation bubble) within the first fluid channel (420),driving a droplet of the second fluid (470) into the third fluid channel(410). Collapses of the bubble causes a back flow of the extruded secondfluid, causing the formation of a narrow “neck” and quickly leading tothe release the droplet (480) into the third fluid channel (410).

Another illustrative embodiment is shown in FIG. 5. The figure shows adevice, which can be a microfluidic device, comprising a first fluidchannel (520) (e.g., a microchannel) containing a first fluid (522), asecond fluid channel (515) (e.g., a microchannel) containing a secondfluid (517), and a third fluid channel (510) (e.g., a microchannel)containing a third fluid (512) where the second fluid is immiscible inthe third fluid and where the second fluid channel and the third fluidchannel are in fluid communication with each other via an opening (530).In some embodiments this opening is in the form of a nozzle. The secondfluid may include particles or cells (516) that may be subsequentlyencapsulated in the generated fluid droplet, and can be in fluidcommunication with the first fluid channel (520) via an aperture (535)or similar structure. A directed energy source (540), for example apulse laser, is directed towards the first fluid channel (520),optionally using a mirror (550), and directed (and optionally focused)into a volume of the first fluid channel (520) using, for example, alens (555). In some embodiments the mirror and/or the lens areconfigured to permit focusing of the directed energy source at differentpositions within the first fluid channel (520). The directed energysource (540) initiates the formation of a transient bubble (560) (e.g.,a cavitation bubble) within the first fluid channel (520), driving adroplet of the second fluid (570) into the third fluid channel (510).Collapse of the bubble causes a back flow of the extruded second fluid,causing the formation of a narrow “neck” and quickly leading to therelease the droplet (580) into the third fluid channel (510).

Another embodiment of the invention is shown in FIG. 6. The figure showsa device, which can be a microfluidic device, comprising a first fluidchannel (620) (e.g., a microchannel) containing a first fluid (622), asecond fluid channel (615) (e.g., a microchannel) containing a secondfluid (617), and a third fluid channel (610) (e.g., a microchannel)containing a third fluid (612) where the second fluid is immiscible inthe third fluid and where the second fluid channel and the third fluidchannel are in fluid communication with each other via an opening (630).In some embodiments this opening is in the form of a nozzle. A flexiblemembrane (635) is interposed between the first fluid channel (620) andthe second fluid channel (615). A directed energy source (640), forexample a pulse laser, is directed towards the first fluid channel(520), optionally using a mirror (650) and focused into a volume of thefirst fluid channel (620) optionally using a lens (655). In someembodiments the mirror and/or the lens are configured to permit focusingof the directed energy source at different positions within the firstfluid channel (620). The directed energy source (640) initiates theformation of a transient bubble (660) (e.g., a cavitation bubble) withinthe first fluid channel (620), which results in an elastic deformationof the flexible membrane (635). This elastic deformation drives adroplet of the second fluid (670) into the third fluid channel (610).Reversal of the elastic deformation following collapse of the bubble(660) results in a back flow of the extruded second fluid, causing theformation of a narrow “neck” and quickly leading to the release thedroplet (680) into the third fluid channel (610). Response time of thisconfiguration can be controlled by the stiffness of the elastic membranein addition to the other parameters discussed above.

Yet another embodiment of the invention is shown in FIG. 7. The figureshows a device, which can be a microfluidic device, comprising a firstfluid channel (720) (e.g., a microchannel) containing a first fluid(720), a second fluid channel (715) (e.g., a microchannel) containing asecond fluid (717), and a third fluid channel (710) (e.g., amicrochannel) containing a third fluid (712) wherein the first, second,and third fluids are immiscible (e.g., by virtue of laminar flow and/orchemical immiscibility). The second fluid may include particles or cells(716) that may be subsequently encapsulated in the generated fluiddroplet. A directed energy source (740), for example a pulse laser, isdirected towards the first fluid channel (720), optionally using amirror (750), and focused into a volume of the first fluid channel(720), optionally using a lens (755). In some embodiments the mirrorand/or the lens are configured to permit focusing of the directed energysource at different positions within the first fluid channel (720). Thedirected energy source (740) initiates the formation of a transientbubble (760) (e.g., a cavitation bubble) within the first fluid channel(720), driving a droplet of the second fluid (770) into the third fluidchannel (710). Collapse of the bubble causes a back flow of the extrudedsecond fluid, causing the formation of a narrow “neck” and quicklyleading to the release the droplet (780) into the third fluid channel(710).

While use of a pulse laser as a directed energy source has been notedabove, it should be noted that other energy sources are suitable for usewith the invention. Alternative directed energy sources includenon-laser, high output optical sources (e.g. focused arc lamps),microwave irradiation, inductive heating, and acoustic energy (e.g.ultrasound).

In certain embodiments, pulsed lasers are preferred energy sources.Lasers are advantageous in that they do not require any electrical ormechanical wiring or interconnects to deliver energy. A laser beam canbe focused to any arbitrary 3D location across a transparent substrate.This eliminates the interfacing problems and facilitates the integrationon standard foundry microfluidic chips.

Illustrative lasers include, but are not limited to nanosecond pulsedlaser with a wavelength, for example, at 532 nm. Microsecond, picosecondor femtosecond pulse lasers, and the like, can also be applied. Incertain embodiments the wavelength of laser can also in the UV, visiblelight, or near infrared.

In certain embodiments the devices or systems comprising the devices canincorporate a monitoring device that characterizes the contents of oneor more of the fluid channels. Data from this monitoring device can betransmitted to a controller, which in turn may be configured to triggerthe directed energy source based on data received from the monitor. Forexample, a fluorescence monitor may by aligned with a fluid channel thatcontains fluorescently labeled cells or particles. When data from themonitor indicates that a cell containing the desired fluorescent labelis aligned with droplet generating mechanism, the controller caninitiate a laser pulse that results in the formation of a droplet thatencapsulates the desired cell. Similarly, absorbance may be used todifferentiate contents of a monitored fluid stream. This arrangementadvantageously permits selection of specific volumes within a fluidchannel that may have unique or desirable contents for transfer to asecond fluid channel for collection or distribution to anotherfunctional area of the device. Monitors are not limited to fluorescenceor absorbance monitors. For example, magnetic monitors, capacitancemonitors, inductance monitors, electrochemical monitors can similarly beused to advantage.

It will be noted that while in certain embodiments, one or more of thefluid streams (e.g., fluid paths) may be confined within physicalchannels (e.g., microchannels), the fluid streams need not beconstrained or separated by a physical barrier/channel wall. In certainembodiments fluid streams can be confined and/or separated, and/ordirected along predetermined paths by variations in thepolarity/hydrophobicity/surface free energy of the surface upon whichthey are disposed (see, e.g., Zhao et al. (2002) Anal. Chem., 74(16):4259-4268), by the use of electrowetting techniques (see, e.g., Chengand Hsiung (2004) Biomedical Microdevices, 6(5): 341-347), byelectrokinetic means, by the use of directed laminar flow (e.g., byadjusting flow rates, and/or stream cross-section, and/or streamviscosity), and the like.

In certain embodiments, the fluid streams are microfluid streams. A“microfluid stream” refers to a stream wherein at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90%, or at least about 95%, or atleast about 98%, or at least about 99%, of the flux or mass of saidfluid stream passes through a cross-sectional area having at least onecharacteristic dimension (e.g., width or diameter) less than 1,000 μm,more preferably less than about 900 μm, or less than about 800 μm, orless than about 700 μm, or less than about 600 μm, or less than about500 μm, or less than about 400 μm, or less than about 300 μm, or lessthan about 250 μm, or less than about 200 μm, or less than about 150 μm,or less than about 100 μm, or less than about 75 μm, or less than about50 μm, or less than about 40 μm, or less than about 30 μm, or less thanabout 20 μm, or less than about 10 μm, or less than about 1 μm. Incertain embodiments the “microfluid stream” refers to a fluid streamcontained within a microfluidic channel.

In certain embodiments one or more of the fluid streams are disposed ina channel or a microchannel. The terms “microfluidic channel” or“microchannel” are used interchangeably and refer to a channel having atleast one characteristic dimension (e.g., width or diameter) less than1,000 μm, more preferably less than about 900 μm, or less than about 800μm, or less than about 700 μm, or less than about 600 μm, or less thanabout 500 μm, or less than about 400 μm, or less than about 300 μm, orless than about 250 μm, or less than about 200 μm, or less than about150 μm, or less than about 100 μm, or less than about 75 μm, or lessthan about 50 μm, or less than about 40 μm, or less than about 30 μm, orless than about 20 μm.

In certain embodiments the methods and devices described herein mayutilize immiscible fluids. In this context, the term “immiscible” whenused with respect to two fluids indicates that the fluids when mixed insome proportion, do not form a solution. Classic immiscible materialsare water and oil. Immiscible fluids, as used herein also include fluidsthat substantially do not form a solution when combined in someproportion. Commonly the materials are substantially immiscible whenthey do not form a solution if combined in equal proportions. In certainembodiments immiscible fluids include fluids that are not significantlysoluble in one another, fluids that do not mix for a period of time dueto physical properties such as density or viscosity, and fluids that donot mix for periods of time due to laminar flow.

In addition, such fluids are not restricted to liquids but may includeliquids and gases. Thus, for example, where the droplets are to beformed comprising an aqueous solvent (such as water) any number oforganic compounds such as carbon tetrachloride, chloroform, cyclohexane,1,2-dichloroethane, dichloromethane, diethyl ether, dimethyl formamide,ethyl acetate, heptane, hexane, methyl-tert-butyl ether pentane,toluene, 2,2,4-trimethylpentane, and the like are contemplated. Variousmutually insoluble solvent systems are well known to those skilled inthe art (see e.g. Table 1). In another example, droplets of aqueousbuffer containing physiologically normal amounts of solute may beproduced in a dense aqueous buffer containing high concentrations ofsucrose. In yet another example, droplets of an aqueous buffercontaining physiologically normal amounts of solute may be produced in asecond aqueous buffer containing physiologically normal amounts ofsolute where the two buffers are segregated by laminar flow. In stillanother example, droplets of a fluid may be produced in a gas such asnitrogen or air.

Table 1 illustrates various solvents that are either miscible orimmiscible in each other. The solvent on left column does not mix withsolvents on right column unless otherwise stated.

Solvents Immiscibility Acetone can be mixed with any of the solventslisted in the column at left Acetonitrile cyclohexane, heptane, hexane,pentane, 2,2,4-trimethylpentane carbon can be mixed with any of thesolvents listed in the column at left except tetrachloride waterchloroform can be mixed with any of the solvents listed in the column atleft except water cyclohexane acetonitrile, dimethyl formamide, dimethylsulfoxide, methanol, water 1,2- can be mixed with any of the solventslisted in the column at left except dichloroethane water dichloromethanecan be mixed with any of the solvents listed in the column at leftexcept water diethyl ether dimethyl sulfoxide, water dimethylcyclohexane, heptane, hexane, pentane, 2,2,4-trimethylpentane, waterformamide dimethyl cyclohexane, heptane, hexane, pentane,2,2,4-trimethylpentane, diethyl solfoxide ether 1,4-dioxane can be mixedwith any of the solvents listed in the column at left ethanol can bemixed with any of the solvents listed in the column at left ethylacetate can be mixed with any of the solvents listed in the column atleft except water heptane acetonitrile, dimethyl formamide, dimethylsulfoxide, methanol, water hexane acetonitrile, dimethyl formamide,dimethyl sulfoxide, methanol, acetic acid, water methanol cyclohexane,heptane, hexane, pentane, 2,2,4-trimethylpentane methyl-tert-butyl canbe mixed with any of the solvents listed in the column at left exceptether water pentane acetonitrile, dimethyl formamide, dimethylsulfoxide, methanol, water, acetic acid 1-propanol can be mixed with anyof the solvents listed in the column at left 2-propanol can be mixedwith any of the solvents listed in the column at left tetrahydrofurancan be mixed with any of the solvents listed in the column at lefttoluene can be mixed with any of the solvents listed in the column atleft except water 2,2,4- acetonitrile, dimethyl formamide, dimethylsulfoxide, methanol, water trimethylpentane water carbon tetrachloride,chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, diethylether, dimethyl formamide, ethyl acetate, heptane, hexane,methyl-tert-butyl ether, pentane, toluene, 2,2,4- trimethylpentane

In certain embodiments the first fluid and second fluid need not beimmiscible in each other. In such embodiments, injected droplets can bekept separate from each other simply by adjusting flow rates in themicrochannels and rate of bubble formation to form separated bubbles.

In various embodiments the droplets generated by the devices and methodsdescribed herein can contain or encapsulate a wide variety of materials.In some embodiments the droplets may contain test samples, cells,organelles, proteins, nucleic acids, enzymes, PCR or other testingreagents, biochemicals, dyes, or particulates (for example polymericmicrospheres, metallic microparticles, or pigments). In still otherembodiments a droplet may encapsulate one or more previously generateddroplets. In addition, the invention need not be limited to aqueousdroplet systems. For example, such droplet generating methods anddevices may be used in nanoparticle coating, where materials in organicsolvents can be used to deposit layers on or encapsulate nanoparticles.

As noted above, in some embodiments an opening in a fluid channel can beconfigured as a nozzle. The depth, inner diameter, and outer diameter ofsuch a nozzle can be optimized to control droplet size, dropletuniformity, mixing at the fluid interface, or a combination of these.

The droplet generation devices described herein may be provided on asubstrate that differs from the material that comprises the fluidchannels. For example, the fluid channels may be fabricated using anelastomeric material that is disposed upon a rigid surface. Suitablefluid channel materials include but are not limited to flexible polymerssuch as PDMS, plastics, and similar materials. Fluid channels may alsobe comprised of nonflexible materials such as rigid plastics, glass,silicon, quartz, metals, and similar material. Suitable substratesinclude but are not limited to transparent substrates such as polymers,plastic, glass, quartz, or other dielectric materials. Other suitablesubstrate materials include but are not limited to nontransparentmaterials such as opaque or translucent plastics, silicon, metal,ceramic, and similar materials.

The parameters described above and in the Examples (e.g., flow rate(s),laser intensity, laser frequency/wavelength, channel dimensions,port/nozzle dimensions, channel wall stiffness, location of cavitationbubble formation, and the like) can be varied to optimize dropletformation and/or droplet/particle/cell encapsulation for a particulardesired application.

There are a number of formats, materials, and size scales that may beused in the construction of the droplet generating devices describedherein and in microfluidic devices that may incorporate them. In someembodiments the droplet generating devices and the connecting fluidchannels are comprised of PDMS (or other polymers), and fabricated usingsoft lithography. PDMS is an attractive material for a variety ofreasons, including but not limited to low cost, optical transparency,ease of molding, and elastomeric character. PDMS also has desirablechemical characteristics, including compatibility with both conventionalsiloxane chemistries and the requirements of cell culture (e.g. lowtoxicity, gas permeability). In an illustrative soft lithography method,a master mold is prepared to form the fluid channel system. This mastermold may be produced by a micromachining process, a photolithographicprocess, or by any number of methods known to those with skill in theart. Such methods include, but are not limited to, wet etching,electron-beam vacuum deposition, photolithography, plasma enhancedchemical vapor deposition, molecular beam epitaxy, reactive ion etching,and/or chemically assisted ion beam milling (Choudhury (1997) TheHandbook of Microlithography, Micromachining, and Microfabrication, Soc.Photo-Optical Instru. Engineer.; Bard & Faulkner, Fundamentals ofMicrofabrication).

Once prepared the master mold is exposed to a pro-polymer, which is thencured to form a patterned replica in PDMS. The replica is removed fromthe master mold, trimmed, and fluid inlets are added where required. Thepolymer replica may be optionally be treated with a plasma (e.g. an O₂plasma) and bonded to a suitable substrate, such as glass. Treatment ofPDMS with O₂ plasma generates a surface that seals tightly andirreversibly when brought into conformal contact with a suitablesubstrate, and has the advantage of generating fluid channel walls thatare negatively charged when used in conjunction with aqueous solutions.These fixed charges support electrokinetic pumping that may be used tomove fluid through the device. While the above described fabrication ofa droplet generating device using PDMS, it should be recognized thatnumerous other materials can be substituted for or used in conjunctionwith this polymer. Examples include, but are not limited to, polyolefinplastomers, perfluoropolyethylene, polyurethane, polyimides, andcross-linked phenol/formaldehyde polymer resins.

In some embodiments single layer devices are contemplated. In otherembodiments multilayer devices are contemplated. For example, amultilayer network of fluid channels may be designed using a commercialCAD program. This design may be converted into a series oftransparencies that is subsequently used as a photolithographic mask tocreate a master mold. PDMS cast against this master mold yields apolymeric replica containing a multilayer network of fluid channels.This PDMS cast can be treated with a plasma and adhered to a substrateas described above.

As noted above, the methods and devices described herein areparticularly suitable for use in microfluidic devices. In someembodiments therefore the fluid channels are microchannels. Suchmicrochannels have characteristic dimensions ranging from about 100nanometers to 1 micron up to about 500 microns. In various embodimentsthe characteristic dimension ranges from about 1, 5, 10, 15, 20, 25, 35,50 or 100 microns up to about 150, 200, 250, 300, or 400 microns. Insome embodiments the characteristic dimension ranges from about 20, 40,or about 50 microns up to about 100, 125, 150, 175, or 200 microns. Invarious embodiments the wall thickness between adjacent fluid channelsranges from about 0.1 micron to about 50 microns, or about 1 micron toabout 50 microns, more typically from about 5 microns to about 40microns. In certain embodiments the wall thickness between adjacentfluid channels ranges from about 5 microns to about 10, 15, 20, or 25microns.

In various embodiments the depth of a fluid channel ranges from 5, 10,15, 20 microns to about 1 mm, 800 microns, 600 microns, 500 microns, 400microns, 300 microns, 200 microns, 150 microns, 100 microns, 80 microns,70 microns, 60 microns, 50 microns, 40 microns, or about 30 microns. Incertain embodiments the depth of a fluid channel ranges from about 10microns to about 60 microns, more preferably from about 20 microns toabout 40 or 50 microns. In some embodiments the fluid channels can beopen; in other embodiments the fluid channels may be covered.

As noted above, some embodiments of the invention include a nozzle.Where a nozzle is present, the nozzle diameter can range from about 0.1micron, or about 1 micron up to about 300 microns, 200 microns, or about100 microns. In certain embodiments the nozzle diameter can range fromabout 5, 10, 15, or 20 microns up to about 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, or about 80 microns. In some embodiments the nozzlediameter ranges from about 1, 5, 10, 15, or 20 microns to about 25, 35,or 40 microns.

In some embodiments the methods and devices described herein cangenerate droplets at a rate ranging from zero droplets/sec, about 2droplets/sec, about 5 droplets/sec, about 10 droplets/sec, about 20droplets/sec, about 50 droplets/sec, about 100 droplets/sec, about 500droplets/sec, or about 1000 droplets/sec, up to about 1,500droplets/sec, about 2,000 droplets/sec, about 4,000 droplets/sec, about6,000 droplets/sec, about 8,000 droplets/sec, about 10,000 droplets/sec,about 20,000 droplets/sec, about 50,000 droplets/sec, and about 100,000droplets/sec.

In various embodiments the devices and methods described herein cangenerate droplets having a substantially continuous volume. Dropletvolume can be controlled to provide volumes ranging from about 0.1 fL,about 1 fL, about 10 fL, and about 100 fL to about 1 microliter, about500 nL, about 100 nL, about 1 nL, about 500 pL or about 200 pL. Incertain embodiments volume control of the droplet ranges from about 1 pLto about 150 pL, about 200 pL, about 250 pL, or about 300 pL.

As indicate above, the microchannel droplet formation/injection devicesdescribed herein can provide a system integrated with other processingmodules on a microfluidic “chip” or in flow through fabrication systemsfor microparticle coating, microparticle drug carrier formulation, andthe like. These uses, however, are merely illustrative and not limiting.

In various embodiments microfluidic that incorporatecomponents/modules/devices that performing the methods described hereincan can manipulate volumes as small as one to several nanoliters.Because the microfluidic reaction volume is close to the size of singlemammalian cells, material loss is minimized in single-cell mRNA analysiswith these devices. The ability to process live cells insidemicrofluidic devices provides a great advantage for the study ofsingle-cell transcriptomes because mRNA is rapidly degraded with celldeath. One illustrative highly integrated microfluidic device, having 26parallel 10 nL reactors for the study of gene expression in single humanembryonic stem cells (hESC) has been reported (Zhong et al. (2008) Labon a Chip, 8: 68-74; Zhong et al. (2008) Curr. Med. Chem., 15:2897-2900) and can be easily modified to intetrate the devices describedherein. Certain illustrative microfluidic devices include systems forobtaining single-cell cDNA including cell capture, mRNAcapture/purification, cDNA synthesis/purification, are performed insidethe device. The present devices and methods offer effective means ofencapsulating and and/or separating individual cells for, e.g., furtherprocessing, in such devices.

Any of a number of approaches can be used to convey the fluids, ormixtures of droplets, particles, cells, etc. along the flow paths and/orchannels of the devices described herein. Such approaches include, butare not limited to gravity flow, syringe pumps, peristaltic pumps,electrokinetic pumps, bubble-driven pumps, and air pressure drivenpumps.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Droplet Generation Driven by Pulse-Laser Induced Cavitation

A pulse laser-driven droplet generation (PLDG) device as shown in FIG. 3was constructed using standard soft lithography techniques. The PLDGdevice had two fluid channels, one filled with water and the other withoil. Both fluid channels were 100 microns in width and 100 microns inheight. The fluid channels were connected with an opening configured asa nozzle, with a neck that was 30 microns in width. Flow rates in thechannels were adjusted to produce a stable oil/water interface.

The actuation of this PLDG device was based on a laser pulse-inducedcavitation bubble, generated when an intense laser pulse was focusedinto the water containing fluid channel. Plasma formation at the focalpoint of the laser pulse generates a rapidly expanding cavitationbubble, as described above. This perturbs the oil/water interface andpushes a droplet of water into the neighboring oil-filled fluid channelto from stable water droplets. The lifetime of this cavitation bubbleranged from tens to hundreds of microseconds in these studies.

To induce cavitation bubbles a Q-switched Nd:YVO4 pulsed laser beam witha wavelength of 532 nm, a 15 nsec pulse width, and a maximum repetitionfrequency of 100 KHz was focused through a 100× objective lens into thePLDG device. Other wavelengths, such as UV, visible, and infrared mayalso be suitable. Droplet generation was captured using a time resolvedimaging system. FIG. 8 shows a series of such images obtained duringdroplet generation. Using corn oil for a continuous oil phase andphosphate buffered saline (PBS) for an aqueous phase, corn oil and PBSflow rates were adjusted to form a stable interface at the nozzleopening (FIG. 8, panel (a)). Cavitation bubble formation is initiatedwithin 1 microsecond of the initiating laser pulse (FIG. 8 panel (b))and reaches maximum size within 3 microseconds, pushing PBS into thecorn oil channel (FIG. 8, panel (c)). The bubble begins to collapseafter 5 microseconds (FIG. 8, panel (d)). As the cavitation bubblecollapses a narrow neck is formed between the PBS fluid channel and theextruded droplet (FIG. 8, panels (d) to (f)). This connection severs dueto hydrodynamic instability (FIG. 8, panel (g)). As a result a 137 pLdroplet was generated using a 100 microjoule laser pulse in about 500microseconds, then transported away by flow through the corn oil channel(FIG. 8, panels (h) and (i)8H).

Example 2 Volume Control of Droplets Generated by PLDG

The volume of PLDG can be controlled can be controlled by adjusting theenergy delivered by the pulse laser, which is a function of laserintensity and pulse duration, the location of the laser excitation, or acombination of the above. Alternatively, the energy of the pulse lasermay be adjusted using a beam polarizer.

FIG. 9 illustrates control of the volume of droplets produced by PLDG byadjusting these parameters. Droplets indicated by FIG. 9, panes (a) to(d), show the effects of varying the laser energy (FIG. 9, panel (a)=100microjoules, panel (b)=90 microjoules, panel (c)=80 microjoules, panel(d)=70 microjoules) at a fixed distance of 47 microns from the nozzles.This produces controlled droplet sizes ranging from about 55 to about 5microns, decreasing with decreasing laser energy.

Control of droplet size is shown in FIG. 9 in panels (e) to (g), wherelaser energy is held constant at 100 microjoules and the distance of thefocus point to the nozzle is adjusted between about 40 microns and about80 microns. Droplet size decreases from about 60 microns to about 25microns as the focus point is moved away from the corn oil/PBSinterface. Using a combination of laser energy and focal point distancefrom the fluid interface droplet volume can be controlled between 1 pLto 150 pL.

Example 3 Consistency of the Size of Droplets Produced by PLDG

Since it is an on demand methodology, PLDG can produce droplets atdifferent frequencies by controlling the interval between laser pulses.FIG. 10 shows the results of continuous droplet generation at differentexcitation intervals ranging from 2 msec (FIG. 10, panel (a)) to 100microseconds (FIG. 10, panel (d)). The flow rate of the fluid channelreceiving the droplets was adjusted to keep the droplets dispersed athigh droplet generation rates.

FIG. 11 shows illustrative e droplets collected at droplet generationfrequencies of 1 kHz (panel (a)) and 10 kHz (panel (b)). Droplet sizewas consistent despite a 10 fold difference in the rate at which thedroplets are formed. FIG. 12 shows results from a similar study, inwhich the interval between laser excitations was set at 2 msec (panel(a)), 500 microseconds (panel (b)), and 100 microseconds (panel (c)).Data collected from droplets generated at 500 microsecond intervals (2kHz) showed a volume variation of 0.689%.

Continuous generation of droplets at different laser excitationintervals is shown in FIG. 14, with excitation intervals at 2 msec(panel (a)), 500 microseconds (panel (b)), and 100 microseconds (panel(c)). Using a pulse interval of 100 microseconds and a laser power of 90microjoules a consistent droplet production rate of 10 kHz can beachieved.

Example 4 Encapsulation in Droplets by PLDG

Since it is an on demand methodology that also permits droplet volumecontrol, PLDG permits the encapsulation of specified contents of a fluidchannel as droplets in a second fluid channel. An example of such anapplication is the encapsulation of a single particle or cell designatedfrom a stream of particles or cells passing through a PLDG device, asdirected by a controller based on data received from a monitor. Such aparticle or cell could be isolated within a droplet of growth media andcarried by a second fluid channel for further characterization.

This is shown in FIG. 13. In FIG. 13, panel (a), particles (indicated bywhite arrows) are shown in s fluid channel of a PLDG device. Generationof the encapsulating droplet is shown in FIG. 13, panel (b). The dropletseen extruding through the nozzle at 250 microseconds from induction ofthe cavitation bubble can be seen to enclose a particle. FIG. 13, panel(c), shows results of a similar study, with continuous capture of cells.Encapsulation of live HeLa cells in this fashion shows high viabilityrates (92.07%). PLDG device reliability has been tested by continuouslyapplying laser pulses at a rate of 10 kHz for one hour, corresponding tothe generation of 3.6 million cavitation bubble generations with noobservable damage to the device.

Droplet generation methods and devices that are particularly suited touse in microfluidic devices have been disclosed. These provide forrapid, on demand droplet generation at rates as high as 100 kHz. Dropletvolume can be adjusted and has been shown to be highly reproducible,with volume differences of less than 1%. The disclosed devices do notutilize mechanical parts, and the use of an externally located directedenergy source (for example a pulse laser) greatly simplifies design ofboth the device and supporting equipment. It should also be noted thatthe efficiency and inherent simplicity of the PLDG approach may haveutility outside of the field of microfluidics. The high rate of dropletproduction and the narrow size distribution of the resulting dropletsindicate that such methods and devices may have utility in thepreparation of emulsions where consistency of the droplet size isparamount. Examples include but are not limited to pharmaceuticals,including vaccine compositions. The high rate of droplet production andthe ability to control the volume of droplets as they are extrudedindicate that such methods and devices may have utility in thedeposition of generated droplets across a fluid/gas interface and ontosolid surfaces, thereby depositing and localizing nonvolatile dropletcontents. Examples of such uses include but are not limited to highresolution printing and generation of microarrays. It should beapparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes. However, where a definition or use of a termin a reference, which is incorporated by reference herein isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply. The terms “comprises” and“comprising” should be interpreted as referring to elements, components,or steps in a non-exclusive manner, indicating that the referencedelements, components, or steps may be present, or utilized, or combinedwith other elements, components, or steps that are not expresslyreferenced.

What is claimed is:
 1. A method for generating droplets in a devicecomprising: providing a first fluid stream comprising a first fluidflowing in a laminar flow; a second fluid stream comprising a secondfluid flowing in a laminar flow adjacent to said first fluid streamwhere said second fluid is a different fluid than said first fluid; andusing a laser to generate a cavitation bubble in the first fluid stream,wherein the cavitation bubble imparts sufficient velocity to a portionof the first fluid so as to extrude a droplet of the first fluid intothe second fluid stream where said droplet is formed as a discretedroplet in said second fluid stream.
 2. The method of claim 1, wherein:said first fluid stream is in a first microfluidic channel; said secondfluid stream is in said second microfluidic channel; and an openingfluidly couples said first microfluidic channel to said secondmicrofluidic channel; and said cavitation bubble imparts sufficientvelocity to a portion of the first fluid so as to extrude a droplet ofthe first fluid across the opening and into the second microfluidicchannel where said droplet is formed as a discrete droplet in saidmicrofluidic channel.
 3. The method of any one of claim 1 or 2, whereinthe first fluid and the second fluid are respectively immiscible to eachother.
 4. The method of claim 1, wherein the cavitation bubble isgenerated by irradiation of a volume of the first fluid using a pulselaser.
 5. The method of claim 4, further comprising selecting at leastone of an intensity, duration, wavelength, and position of theirradiation produced by said pulse laser to thereby produce a desiredvolume of the droplet.
 6. The method of claim 4, wherein the first fluidand the second fluid are respectively immiscible to each other.
 7. Themethod of claim 6, wherein the first fluid comprises an aqueous fluid.8. The method of claim 7, wherein the second fluid comprises an oil oran organic solvent.
 9. The method of claim 4, wherein the irradiation isinitiated by a controller controlling said pulse laser.
 10. The methodof claim 9, further comprising a step of selecting at least one oftiming of occurrence of pulses emitted by the pulse laser, frequency ofoccurrence of pulses emitted by the pulse laser, wavelength of pulsesemitted by the pulse laser, energy of pulses emitted by the pulse laser,and aiming or location of pulses emitted by the pulse laser.
 11. Themethod of claim 9, further comprising monitoring via a monitor thesecond fluid path and transmitting data generated by such monitoring tosaid controller.
 12. A device for generating droplets comprising: afirst microfluidic channel containing a first fluid; a secondmicrofluidic channel containing a second fluid that is different thansaid first fluid; an opening between the first microfluidic channel andthe second microfluidic channel; a laser; and a controller coupled tosaid laser and configured to operate said laser to induce temporaryformation of one or more cavitation bubbles in the first fluid in saidfirst microfluidic channel to form a bubble in the first fluid effectiveto thereby extrude a droplet of the first fluid through the opening intothe second microfluidic channel where said droplet is formed as adiscrete droplet in said second fluid.
 13. The device of claim 12,wherein the controller is configured to adjust volume of the droplet asa function of at least one of timing of occurrence of pulses emitted bythe laser, frequency of occurrence of pulses emitted by the laser,wavelength of pulses emitted by the laser, energy of pulses emitted bythe laser, and aiming or location of pulses emitted by the laser. 14.The method of claim 12, wherein the first fluid and the second fluid arerespectively immiscible to each other.
 15. The device of claim 12,wherein said laser is a pulse laser configured to produce asubstantially continuous volume tuning of droplet size ranging fromabout 0.1 fL to about 1μL.
 16. The device of claim 12, wherein saidlaser is a pulse laser configured to provide on-demand dropletgeneration at a speed of greater than about 1,000 droplets/sec.